Stuck  Like  Glu:  Glutamyl-­‐tRNA  Reductase  (GluTR)  East  Brunswick  High  School  

Abstract  Glutamyl-­‐tRNA   reductase   (GluTR)   catalyzes   the  reducBon   of   glutamyl-­‐tRNAGlu   into   glutamate-­‐1-­‐semialdehyde   (GSA).  We   explored   the   structure  and   funcBon   of  Arabidopsis   thaliana   GluTR   and  its   sBmulator   protein,   GluTR   binding   protein  (GluBP),  to  infer  the  role  of  GluTR  in  the  aquaBc  duckweed  plant,  Landol0a  punctata.  

IntroducBon  Tetrapyrroles  are  a  class  of  chemical  compounds  useful   in  many  biological  processes.  Examples  of  tetrapyrroles   include   chlorophyll,   heme,   and  phytochromobilin.  5-­‐Aminolevulinic  acid   (ALA)   is  the   universal   precursor   for   tetrapyrrole  biosynthesis.  In  plants,  algae,  and  most  bacteria,  ALA   is   produced   in   a   2-­‐step   mechanism   (see  Figure   1).   Stroma-­‐localized   GluTR   catalyzes   the  first,  rate-­‐limiBng  step  of  that  mechanism.  

Informally,  GluTR  acBvity  divides   into  two  steps:(1)   glutamate   is   extracted   from   the   glutamyl-­‐tRNAGlu   and   converted   into   a   thioester  intermediate   and   (2)   the   thioester   intermediate  is  reduced  into  GSA.  Thylakoid  membrane-­‐bound  GluBP   sBmulates   GluTR   catalyBc   acBvity   by  causing   a   conformaBonal   change   that   favors  electron  transfer.  

Methods  Using   BLAST   analysis,   students   inferred   the  funcBon   of   over   100   cDNA   sequences   from   L.  punctata.   We   selected   a   sequence   (clone  04SH1.14)  coding  for  part  of  a  domain  on  GluTR  to   further   invesBgate.   AXer   using   similar  sequences  to  infer  the  protein  sequence  that  the  transcript  encoded,  we   researched   the  protein’s  structure,  funcBon,  and  applicaBons.  

Discussion  GluTR   is   a   homodimer,   in   which   each   subunit  contains   3   domains:   an   N-­‐terminal   catalyBc  domain  where  glutamyl-­‐tRNAGlu  and  GluBP  bind,  an   NADPH-­‐binding   domain,   and   a   C-­‐terminal  dimerizaBon   domain   that   holds   the   dimer  together.    

Figure  1:  5-­‐Aminolevulinic  Acid  (ALA)  is  produced  in  a  2-­‐step  mechanism.  First,  glutamyl-­‐tRNAGlu   is   reduced   into  GSA  by  GluTR   in  a   reacBon  which  GluBP   sBmulates.   Then,   GSAM   isomerizes   GSA   into   ALA,   which   can   be  converted  into  tetrapyrroles.  

Figure   2:   GluTR   also   funcBons   in   2   steps.   In   the   first   step,   it   extracts  glutamate   from   glutamyl-­‐tRNAGlu,   creaBng   a   thioester   intermediate  whose  thioester  group  is  labeled  in  red.  Then,  the  thioester  intermediate  is  reduced  into  GSA.    

Figure   3:   GluTR   in   complex   with   its   sBmulator   protein,   GluBP.   Both  proteins   are   homodimers.   GluTR   monomers   each   have   3   domains   and  GluBP  monomers  each  have  two  domains.  

Discussion  The  reacBon  starts  when  glutamyl-­‐tRNAGlu  binds  to   the   catalyBc   domain   on   each   monomer   of  GluTR.   The   negaBvely   charged   tRNAGlu   is  a]racted   to   a   posiBve   region   on   GluTR.   A  conserved   arginine   (Arg415)   also   recognizes   the  tRNAGlu  and  another  conserved  arginine  (Arg146)  recognizes  the  glutamate.    

Figure  4:  NegaBvely  charged  tRNAGlu  (purple)  superimposed  on  posiBvely  charged  region  where  it  binds  to  on  GluTR  (blue)  

Then,   Cys144   separates   the   glutamate   from   the  tRNAGlu  by  breaking  the  aminoacyl  bond  between  glutamate  and  the  tRNAGlu,  forming  the  thioester  intermediate   at   the   GluTR   catalyBc   domains.  Meanwhile,  NADPH  binds  to  the  NADPH-­‐binding  domains.  Next,  GluBP  binds   to  GluTR,   stabilizing  the   V-­‐structure   of   GluTR   and   driving   a  conformaBonal   change   that   twists   the   spinal   α-­‐helix   on   each   GluTR   subunit.   This   swings   the  NADPH-­‐binding   domains   toward   the   catalyBc  domains,  making  it  easier  for  NADPH  to  transfer  electrons  to  the  thioester  intermediate.  

Figure   5:   Thioester   intermediate   (yellow   sBck)   receiving   electrons   from  NADPH  (other  sBck  model).  

Once  NADPH  reduces  the  thioester  intermediate  into   GSA,   the   product   is   then   shu]led   out  towards  the  center  of  the  complex  as  a  result  of  GSA   interacBons   with   Gly101,   Cys144,   Arg146,  and  His193  on  GluTR.    

AXer   being   channeled,   GSA   is   held   in   an   exit  pocket   on   GluTR   consisBng   of:   His105,   Glu148,  Phe183,   and  Asp202.  GSA   is   shielded   by   Lys271  on   GluBP   to   prevent   it   from   escaping.   When  GluBP   detaches,   GSA-­‐2,1-­‐aminomutase   (GSAM),  the   next   enzyme   in   the   2-­‐step   ALA   synthesis  mechanism,   binds   to   a   similar   region   on   GluTR.  Since   the   channel   is   no   longer   shielded,   GSAM  accepts  GSA  and  isomerizes  it  into  ALA.    

Figure  6:  Review  of  ALA  synthesis  pathway  

Conclusion  

Figure   7:   (a)   Coupled   enzyme   assay   reveals   3-­‐fold   increase   in   GluTR  acBvity   by   GluBP   binding   (b)   Coupled   enzyme   assay   reveals   heme   is   a  negaBve   allosteric   regulator   of   GluTR   (c)   Northern   blot   analysis   shows  gene  coding  for  GluTR  is  only  expressed  during  light  exposure  

Many   other   molecules   and   condiBons   affect  GluTR   acBvity.   Coupled   enzyme   assays   reveal  that  GluBP  increases  GluTR  acBvity  3-­‐fold.  Heme  negaBvely   allosterically   regulates   GluTR,   which  allows   cells   to   conserve   both   energy   and  resources  by  prevenBng  the  producBon  of  excess  ALA.   Northern   blot   analysis   indicates   that  increasing  light  and  temperature  increases  GluTR  producBon.   Therefore,   GluTR   is   most   acBve   in  the   presence   of  GluBP,   low   heme   content,   high  temperature,  and  increased  light  exposure.    

A   B  

C  

References  h]p://www.ncbi.nlm.nih.gov/pmc/arBcles/PMC402005,  h]p://www.ncbi.nlm.nih.gov/pmc/arBcles/PMC125327/,  h]p://www.jbc.org/content/274/43/30679.long,  h]p://www.ncbi.nlm.nih.gov/pubmed/15757895,  h]p://www.ncbi.nlm.nih.gov/pubmed/22180625,  h]p://www.plantcell.org/content/6/2/265.full.pdf  

Step  1:  Glu  ExtracBon   Step  2:  ReducBon  

L   L   L   L  D   D  glutamyl-­‐tRNAGlu  

GSA  

ALA  

Tetrapyrroles  (chlorophyll,  heme,  etc.)  

GSAM  

GluTR  GluBP